CDNA and gene for human angiogenin (angiogenesis factor) and methods of expression

ABSTRACT

DNA sequences encoding a protein having angiogenic activity are disclosed. Expression vectors containing these sequences are introduced into host cells and direct the production of the angiogenic protein. Proteins produced according to the invention are useful in the diagnosis of malignancies, for promoting wound healing, and for other diagnositic and therapeutic purposes.

This application is a continuation-in-part of our co-pending applicationU.S. Ser. No. 770,657 filed Aug. 28, 1985, which is hereby incorporatedby reference.

FIELD OF THE INVENTION

This invention relates to protein production through recombinant DNAtechniques. More particularly, it relates to DNA sequences encodingproteins having angiogenic activity and to methods of expressing thosesequences.

BACKGROUND ART

Angiogenesis, the process of developing a hemovascular network, isessential for the growth of solid tumors and is a component of normalwound healing and growth processes. It has also been implicated in thepathophysiology of atherogenesis, arthritis, and diabetic retinopathy.It is characterized by the directed growth of new capillaries toward aspecific stimulus. This growth, mediated by the migration of endothelialcells, may proceed independently of endothelial cell mitosis.

The molecular messengers responsible for the process of angiogenesishave long been sought. Greenblatt and Shubik (J. Natl. Cancer Inst. 41:111-124, 1968) concluded that tumor-induced neovascularization ismediated by a diffusible substance. Subsequently, a variety of solublemediators have been implicated in the induction of neovascularization.These include prostaglandins (Auerbach, in Lymphokines, Pick and Landy,eds., 69-88, Academic Press, New York, 1981), human urokinase (Berman etal., Invest Opthalm. Vis. Sci. 22: 191-199, 1982), copper (Raju et al.,J. Natl. Cancer Inst. 69: 1183-1188, 1982), and various "angiogenesisfactors".

Angiogenesis factors have been derived from tumor cells, wound fluid(Banda et al., Proc. Natl. Acad. Sci USA 79: 7773-7777, 1982; Banda etal., U.S. Pat. No. 4,503,038), and retinal cells (D'Amore, Proc. Natl.Acad. Sci. USA 78: 3068-3072, 1981). Tumor-derived angiogenesis factorshave in general been poorly characterized. Folkman et al. (J. Exp. Med.133: 275-288, 1971) isolated tumor angiogenesis factor from the Walker256 rat ascites tumor. The factor was mitogenic for capillaryendothelial cells and was inactivated by RNase. Tuan et al.(Biochemistry 12: 3159-3165, 1973) found mitogenic and angiogenicactivity in the nonhistone proteins of the Walker 256 tumor. The activefraction was a mixture of proteins and carbohydrate. A variety of animaland human tumors have been shown to produce angiogenesis factor(s)(Phillips and Kumar, Int. J. Cancer 23: 82-88, 1979) but the chemicalnature of the factor(s) was not determined. A low molecular weightnon-protein component from Walker 256 tumors has also been shown to beangiogenic and mitogenic (Weiss et al., Br. J. Cancer 40: 493-496,1979). An angiogenesis factor with a molecular weight of 400-800 daltonswas purified to homogeneity by Fenselau et al. (J. Biol. Chem. 256:9605-9611, 1981), but it was not further characterized. Human lung tumorcells have been shown to secrete an angiogenesis factor comprising ahigh molecular weight carrier and a low molecular weight, possiblynon-protein, active component (Kumar et al., Int. J. Cancer 32: 461-464,1983). Vallee et al. (Experientia. 41: 1-15, 1985) found angiogenicactivity associated with three fractions from Walker 256 tumors. Tolbertet al. (U.S. Pat. No. 4,229,531) disclose the production of angiogenesisfactor from the human adenocarcinoma cell line HT-29, but the materialwas only partially purified and was not chemically characterized.Isolation of genes responsible for the production of angiogenesisfactors has not heretofore been reported at least in part due to thelack of purity and characterization of the factors.

Isolation of angiogenesis factors has employed high performance liquidchromatography (Banda et al., ibid); solvent extraction (Folkman et al.,ibid); chromatography on silica gel (Fenselau et al., ibid), DEAEcellulose (Weiss et al., ibid), or Sephadex (Tuan et al., ibid); andaffinity chromatography (Weiss et al., ibid).

Recently, Vallee et al. (U.S. patent application Ser. No. 724,088, filedApr. 17, 1985, and U.S. Ser. No. 778,387, filed concurrently with thisapplication, both of which are hereby incorporated by reference) havepurufied an angiogenic protein from a human adenocarcinoma cell line.The purified protein, known as angiogenin, was chemically characterizedand its amino acid sequence determined.

Because angiogenesis factors play an important role in wound healing(Rettura et al., FASEB Abstract #4309, 61st Annual Meeting, Chicago,1977) and may find applicability in the development of screening testsfor malignancies (Klagsburn et al., Cancer Res. 36: 110-114, 1976; andBrem et al., Science 195: 880-881, 1977), it would clearly beadvantageous to produce angiogenic proteins in sufficient quantities topermit their application in therapy and diagnosis. The techniques ofgenetic engineering are ideally suited to increase production levels ofthese proteins. The cloning of genes encoding angiogenic proteins is anecessary first step in such a large-scale production.

Furthermore, it may in some instances be desirable to obtain theseproteins from non-tumor cells, such as in the case of humantherapeutics, where contamination with certain tumor products would beunacceptable. This invention therefore provides for the production ofangiogenic proteins in non-tumor cells using recombinant DNA techniques.

DISCLOSURE OF THE INVENTION

Briefly stated, the present invention discloses a DNA sequence encodinga protein having angiogenic activity. A DNA sequence encodingangiogenin, or a protein having substantially the same biologicalactivity as angiogenin, is also disclosed. The DNA sequences may beobtained from cDNA or genomic DNA, or may be prepared by DNA synthesistechniques.

The invention further discloses vectors comprising a DNA sequenceencoding a protein having angiogenic activity. Vectors comprising a DNAsequence encoding a protein having substantially the same biologicalactivity as angiogenin are also disclosed. The vectors further comprisea promoter sequence upstream of and operably linked to the DNA sequence.In general, the vectors will also contain a selectable marker, and,depending on the host cell used, may contain such elements as regulatorysequences, polyadenylation signals, enhancers, and RNA splice sites.

An additional aspect of the present invention discloses cellstransfected or transformed to produce a protein having angiogenicactivity. Cells transfected or transformed to produce a protein havingsubstantially the same biological activity as angiogenin are alsodisclosed. The cells are transfected or transformed to contain anexpression vector comprising a DNA sequence encoding a protein havingangiogenic activity.

A further aspect of the present invention discloses a method forproducing a protein having angiogenic activity. The method comprises (a)introducing into a host cell a vector comprising a DNA sequence encodinga protein having angiogenic activity; (b) growing the host cell in anappropriate medium; and (c) isolating the protein product encoded by theDNA sequence and produced by the host cell. A method for producing aprotein having substantially the same biological activity as angiogeninis also disclosed. The proteins produced by these methods are alsodisclosed.

Other aspects of the invention will become evident upon reference to thedetailed description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the amino acid sequence of angiogenin purified fromhuman adenocarcinoma HT-29 cells.

FIG. 2 illustrates the strategy used for sequencing the angiogenin cDNAand genomic clones. The top portion refers to the cDNA and the bottomportion to the genomic DNA. Solid bars indicate the coding regions,arrows indicate the fragments sequenced. The locations and directions ofthe three Alu sequences are indicated by large hatched arrows.

FIG. 3 illustrates a portion of the sequence of the genomic DNA insertin λHAG1. The cDNA insert of pHAG1 corresponds to nucleotides 106 to 731of the genomic DNA, with a substitution at nucleotide 252.

FIG. 4 illustrates the construction of the mammalian cell expressionvector pHAGF-MT-DHFR.

FIGS. 5 and 6 illustrate the construction of the yeast expression vectorpYAGF.

BEST MODE FOR CARRYING OUT THE INVENTION

Prior to setting forth the invention, it may be helpful to definecertain terms to be used hereinafter.

Biological activity is a function or set of functions performed by amolecule in a biological context (i.e., in an organism or an in vitrofacsimile). For angiogenin, biological activity is characterized by itsangiogenic activity.

Angiogenic activity is the chemical stimulation of hemovasculardevelopment in tissue. It is generally associated with diffusiblesubstances produced by a variety of cell types. Angiogenic activity maybe characterized by a positive response in the chick embryochorioallantoic membrane assay (Knighton et al., Br. J. Cancer 35:347-356, 1977) and/or the rabbit cornea implant assay (Langer andFolkman, Nature 263: 797-800, 1976).

DNA construct is a DNA molecule, or a clone of such a molecule, whichhas been modified by human intervention to contain segments of DNA whichare combined and juxtaposed in a manner which would not otherwise existin nature.

Angiogenic proteins are produced by a variety of cell types, includingtumor cells and retinal cells. Until recently, these proteins have notbeen obtained in sufficient purity to permit their chemical and physicalcharacterization. Through the application of a novel multi-steppurification procedure, an exemplary angiogenic protein, hereinafterangiogenin, has been purified from culture media of a human tumor cellline. Determination of the protein sequence has allowed the isolation ofcorresponding DNA sequences and the expression of these sequencesthrough recombinant DNA techniques.

The isolation of angiogenic proteins is based on the fractionation ofconditioned cell media by ion exchange chromatography, followed by highperformance liquid chromatography.

Although tumor cells are the preferred source of an angiogenic proteinaccording to the present invention, other types of cells, notablyretinal cells, are known to produce angiogenesis factors. A particularlypreferred cell line is the human adenocarcinoma cell line HT-29 (Foghand Trempe, in Human Tumor Cells in Vitro, Fogh, ed., 115-160, Plenum,New York, 1975). HT-29 isolates have been deposited with American TypeCulture Collection under accession numbers HTB38 and CRL8905. The cellsmay be cultured according to known methods, e.g. as monolayer culturesin Dulbecco's modified Eagle's medium or other suitable media. Apreferred medium is Dulbecco's modified Eagle's medium supplemented with2 mM L-glutamine and 5% heat inactivated fetal bovine serum (DME/5). Themedium is changed periodically and cells are subcultured according toknown procedures.

To facilitate isolation of angiogenic protein(s) from the cell medium,it is preferred that the cells be transferred to a serum freemaintenance medium once they have reached confluent growth. A preferredmaintenance medium is DME/5 without serum but containing L-glutamine ata concentration of 5 mM.

Medium in which cells have been cultured or maintained, known asconditioned medium, is then removed from the cells and preferablyfiltered to remove cell debris, then treated to remove high molecularweight proteins. A preferred method of treatment of acidification, e.g.by the addition of glacial acetic acid to a concentration of 5% (v/v),followed by centrifugation. It may also be desirable to concentrate thefiltered, acidified medium prior to further purification steps.

The filtered treated medium is then chromatographed on a cation exchangematrix, such as carboxymethyl cellulose (CM cellulose). It is preferredthat the treated, conditioned medium be lyophilized, reconstituted in0.1M sodium phosphate buffer pH 6.6, and applied to the matrix. Undersuch conditions, the angiogenesis factor(s) will bind to the matrix andmay be eluted with the same buffer containing 1M NaCl.

The eluate from the cation exchange matrix is further fractionated byreversed-phase high performance liquid chromatography. The eluate islyophilized, reconstituted in a suitable solvent, such as 0.1%trifluoroacetic acid (TFA) in water, and eluted by applying a gradientof a second solvent to the column. A linear gradient ofisopropanol/acetonitrile/water (5:5:4 v/v/v) containing 0.08% TFA ispreferred. Material eluted from the HPLC column may then be dialyzed toremove the solvent, lyophilized, and reconstituted.

The reconstituted HPLC column eluate is then assayed for angiogenicactivity to identify the active fraction(s). Several assays forangiogenic activity are well known in the art, including the chickembryo chorioallantoic membrane assay (Knighton et al., Br. J. Cancer35: 347-356, 1977) and the cornea implant assay (Langer and Folkman,Nature 263: 797-800, 1976).

When HT-29 cells are employed as the starting material, two activefractions are obtained from the HPLC column. One fraction contains amajor protein component of M_(r) ˜16,000 and lesser amounts of a M_(r)˜14,000 species. The second fraction contains a single protein speciesof M_(r) ˜14,000, which has been designated angiogenin. On furtheranalysis, angiogenin was found to have an isoelectric point greater than9.5 and a molecular weight of approximately 14,193 daltons by amino acidsequence analysis. Surprisingly, in contrast to most previouslydescribed angiogenesis factors, angiogenin is not mitogenic inconventional assays. The amino acid sequence of angiogenin was found tobe 35% homologous to the pancreatic ribonucleases.

When an angiogenic protein has been obtained in substantially pure form,its amino acid sequence is determined by known methods, for example,Edman degradation (Edman and Begg, Eur. J. Biochem. 1: 80-91, 1967). Itis not necessary to determine the entire amino acid sequence.Preferably, a sequence of at least 5-10 amino acids will be determined.

From the amino acid sequence, a DNA probe is designed. Generally, itwill be necessary to design a family of probes corresponding to all ofthe possible DNA sequences encoding the amino acid sequence. It ispreferred that such a probe be at least 14 nucleotides in length inorder to minimize false positive signals when screening DNA clones.Suitable probes may be synthesized by known methods (for review, seeItakura, in Trends in Biochemical Science, Elsevier Biochemical Press,1982) or purchased from commercial suppliers.

cDNA (complementary DNA) and/or genomic DNA libraries are then preparedand screened with the probe(s) using conventional hybridizationtechniques. Techniques for preparing such libraries are well known inthe art (see, for example, Lawn et al., Cell 15: 1157-1174, 1978; andMichelson et al., Proc. Natl. Acad. Sci. USA 80: 472-476, 1983). Cloneswhich hybridize to the probe(s) are then selected and sequenced.

Alternatively, if a sufficient quantity of pure angiogenic protein isobtained, it may be used to prepare an antibody, and the antibody inturn used to screen an expression cDNA library (Young and Davis, Pro.Natl. Acad. Sci. USA 80: 1194-1198, 1983).

If a full length cDNA clone is obtained, it may be inserted directlyinto an expression vector for use in producing the angiogenic protein.Lacking a full length cDNA clone, the remaining coding sequence may beobtained by several methods, and a full length coding sequence may thenbe constructed. A cDNA clone may be used as a probe to screen additionalcDNA libraries or to screen genomic DNA libraries. If the amino acidsequence of the protein is known, the missing material may besynthesized and joined to the cDNA and/or genomic DNA fragments toconstruct a complete coding sequence. Under some circumstances, it ispreferred that the coding sequence further encode a leader peptide inorder to facilitate proper processing and secretion of the angiogenicprotein produced according to the present invention. The leader peptidemay be that of the angiogenic peptide itself, or a heterologous leaderpeptide which functions in the particular host cell.

When a full length DNA sequence encoding an angiogenic protein has beenobtained, it is then inserted into a suitable expression vector.Expression vectors for use in carrying out the present invention willfurther comprise a promoter operably linked to the DNA sequence encodingthe angiogenic protein. In some instances it is preferred thatexpression vectors further comprise an origin of replication, as well assequences which regulate and/or enhance expression levels, depending onthe host cell selected. Suitable expression vectors may be derived fromplasmids or viruses, or may contain elements of both.

Preferred prokaryotic hosts for use in carrying out the presentinvention are strains of the bacteria Escherichia coli, althoughBecillus and other genera are also useful. Techniques for transformingthese hosts and expressing foreign genes cloned in them are well knownin the art (see, for example, Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, 1982). Vectors usedfor expressing foreign genes in bacterial hosts will generally contain aselectable marker, such as a gene for antibiotic resistance, and apromoter which functions in the host cell. Appropriate promoters includethe trp (Nichols and Yanofsky, Meth. in Enzymology 101: 155, 1983) lac(Casadaban et al., J. Bact. 143: 971-980, 1980) and phage λ promotersystems. Plasmids useful for transforming bacteria include pBR322(Bolivar, et al., Gene 2: 95-113, 1977), the pUC plasmids (Messing,Meth. in Enzymology 101: 20-77, 1983; and Vieira and Messing, Gene 19:259-268, 1982), pCQV2 (Queen, J. Mol. Appl. Genet. 2: 1-10, 1983), andderivatives thereof.

Eukaryotic microorganisms, such as the yeast Saccharomyces cerevisiae,may also be used as host cells. Techniques for transforming yeast aredescribed by Beggs (Nature 275: 104-108, 1978). Expression vectors foruse in yeast include YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76:1035-1039, 1979). YEp13 (Broach et al., Gene 8: 121-133, 1979), pJDB248and pJDB219 (Beggs, ibid), and derivatives thereof. Such vectors willgenerally comprise a selectable marker, such as the nutritional markerTRP, which allows selection in a host strain carrying a trpl mutation.Preferred promoters for use in yeast expression vectors includepromoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem.255: 12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1:419-434, 1982) or alcohol dehydrogenase genes (Young et al., in GeneticEngineering of Microorganisms for Chemicals, Hollaender et al., eds., p.335, Plenum, New York, 1982; and Ammerer, Meth. in Enzymology 101:192-201, 1983). To facilitate purification of an angiogenic proteinproduced in a yeast transformant and obtain proper disulphide bondformation, a signal sequence, preferably from a yeast gene encoding asecreted protein, may be joined to the coding sequence for theangiogenic protein. A particularly preferred signal sequence is thepre-pro region of the MFαl gene (Kurjan and Herskowitz, Cell 30:933-943, 1982).

Higher eukaryotic cells may also serve as host cells in carrying out thepresent invention. Cultured mammalian cells are preferred. Expressionvectors for use in mammalian cells will comprise a promoter capable ofdirecting the transcription of a foreign gene introduced into amammalian cell. A particularly preferred promoter is the mousemetallothionein-1 (MT-1) promoter (Palmiter et al., Science 222:809-814, 1983). Also contained in the expression vectors is apolyadenylation signal, located downstream of the insertion site. Thepolyadenylation signal may be that of the cloned angiogenic proteingene, or may be derived from a heterologous gene.

Cloned gene sequences may then be introduced into cultured mammaliancells by, for example, calcium phosphate-mediated transfection (Wigleret al., Cell 14: 725, 1978; Coraro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52: 456, 1973). A precipitateis formed of the DNA and calcium phosphate, and this precipitate isapplied to the cells. Some of the cells take up the DNA and maintain itinside the cell for several days. A small fraction of these cells(typically 10⁻⁴) integrate the DNA into the genome. In order to identifythese integrants, a gene that confers a selectable phenotype (aselectable marker) is generally introduced into the cells along with thegene of interest. Preferred selectable markers include genes that conferresistance to drugs, such as neomycin, hygromycin, and methotrexate.Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid.

The copy marker of the integrated gene sequence may be increased throughamplification by using certain selectable markers (e.g., dihydrofolatereductase, which confers resistance to methotrexate). The selectablemarker is introduced into the cells along with the gene of interest, anddrug selection is applied. The drug concentration is then increased in astep-wise manner, with selection of resistant cells at each step. Byselecting for increased copy number of cloned sequences, expressionlevels may be substantially increased.

Angiogenic proteins produced according to the present invention may bepurified form the host cells or cell media by cation exchangechromatography and high performance liquid chromatography as describedabove.

It will be appreciated that other angiogenic proteins may be isolated bythe above process. Different cell lines may be expected to produceangiogenic proteins having different physical properties. Additionally,variations may exist due to genetic polymorphisms or cell-mediatedmodifications of the proteins or their precursors. Furthermore, theamino acid sequence of an angiogenic protein may be modified by genetictechniques to produce proteins with altered biological activities. Forexample, based on the homology between antiogenin and ribonuclease, thecys residues at positions 26, 39, 57, 81, 92 and 107, and histidines atpositions 13 and 114, and the lysine at position 40 are preferred sitesfor replacement by other amino acids by site specific mutagenesis(Zoller et al., Manual for Advanced Techniques in Molecular CloningCourse, Cold Spring Harbor Laboratory, 1983). The resultant DNA sequencewill encode a protein having substantially the same amino acid sequenceas angiogenin, but exhibiting a higher or lower level of angiogenicactivity. An increase in the biological activity could permit the use oflower dosage levels. A molecule having reduced angiogenic activity or noangiogenic activity, but retaining certain structural features, couldstill bind receptors on endothelial or other cells and, by blocking thesite of action, form an antagonist to the action of the natural protein,resulting in an approach to the treatment of angiogenesis-relateddisease states. Such proteins are within the scope of the presentinvention.

Angiogenic proteins produced according to the present invention may beused to produce therapeutic or diagnostic compositions by combining themwith suitable carriers. The therapeutic compositions may be used topromote the development of a hemovascular network in a mammal, forexample, to induce collateral circulation following a heart attack, orto promote wound healing, for example in joints or other locations.Preferably, the therapeutic compositions according to the presentinvention will be administered intravenously or by direct topicalapplication to the wound site. For example, if injury occurs to themeniscus of the knee or shoulder as frequently occurs in sports-relatedinjuries or osteoarthritis, injection of angiogenic proteins at the siteof the injury may promote healing of torn or traumatized fibrocartilagematerial. Effective doses will vary according to the severity of thecondition and the target tissue. Furthermore, angiogenic proteins havediagnostic applications in screening for the presence of malignancies,either by using the protein to assay for the presence of antibodies orto produce antibodies for use as immunodiagnostic reagents. A diagnosticcomposition containing the protein may be incubated with a biologicalsample under conditions suitable for the formation of anantigen-antibody complex. The formation of the complex (i.e., thepresence of antibodies in the sample) is then detected. Techniques forsuch assays are well known in the art, e.g. the enzyme linkedimmunosorbent assay (Voller et al., The Enzyme Linked ImmunosorbentAssay, Dynatech Laboratories, Inc., 1979) or the Western blot assay(see, for example, Towbin et al., Proc. Natl Acad. Sci. USA 76: 4350,1979). Similarly, a diagnostic composition comprising an antibodyagainst an angiogenic protein may be used to assay for the presence ofthe protein in a biological sample. The angiogenic proteins may also beused to develop angiogenesis inhibitors which may be useful in thetreatment of disorders associated with angiogenesis. Recombinant DNAprovides a superior method for the production of these proteins in thequantities needed for these applications.

EXPERIMENTAL Materials and Methods

Restriction endonucleases, T4 DNA ligase, T4 kinase, alkalinephosphatase, endonuclease Bal 31 and Klenow fragment of DNA polymerase I(E. coli) were purchased from Bethesda Research Laboratories or NewEngland Biolabs. Reverse transcriptase (avain myeloma virus) wasobtained from Seikagaku U.S.A., Inc. Dideoxynucleotide triphosphates,deoxynucleotide triphosphates, pBR322 and pUC13 were purchased from P-LBiochemicals. Universal primers (hepta decamer) for dideoxy sequencingwere purchased from New England Biolabs, and the [α-³² P]dATP, [γ³²P]ATP, and [³⁵ S]dATPαS were obtained from Amersham.

EXAMPLE 1 Isolation of cDNA and Genomic Sequences Encoding Angiogenin

A human cDNA library was prepared from human liver poly(A)-mRNAemploying plasmid pUC13 as a cloning vector (Maniatis et al., ibid).This plasmid was previously tailed with G's at its PstI site (Michelson& Orkin, 1982). A mixture of 26 synthetic oligonucleotides[CCCTGAGGCTTAGC(A/G)TC(A/G)TA(A/G)TG(C/T)TG] was purchased from P-LBiochemicals and employed as a hybridization probe. The nucleotidemixture is complementary to nucleotide sequences that code forGln-His-Tyr-Asp-Ala-Lys-Pro-Gln-Gly. This sequence is present in theamino-terminal region of human angiogenin isolated from the colonadenocarcinoma cell line HT-29 (see FIG. 1). The nucleotide mixture wasradiolabeled with T4 kinase and [³² P]ATP to a specific activity ofapproximately 3×10⁸ cpm/μg and employed for the screening of 350,000transformants from the liver library by the method of Wallace et al.(Nucleic Acids Res. 9: 879-894, 1981). Seven recombinant plasmids thathybridized strongly with the probe were isolated and purified by cesiumchloride gradient centrifugation. The DNA inserts in each of thepositive clones were digested with various restriction enzymes andanalyzed by polyacrylamide gel electrophoresis. Their sequence wasdetermined by the chemical degradation method of Maxam & Gilbert (Meth.in Enzymology 65: 499-560, 1980). Each sequence was determined two ormore times, and greater than 85% of the sequence was determined on bothstrands.

The plasmid containing the largest cDNA insert (pHAG1) was thensequenced by the method of Maxam & Gilbert (ibid) according to thestrategy shown in the top of FIG. 2. This cDNA insert contained 697nucleotides and included 12 G's at the 5' end, a short noncodingsequence, a leader sequence coding for a signal peptide of 24 (or 22)amino acids, 369 nucleotides coding for the mature protein of 123 aminoacids, a stop codon, 175 nucleotides of 3' noncoding sequence, a poly(A)tail of 36 nucleotides, and 23 C's on the 3' end. The cDNA insertcorresponds to nucleotides 106 to 731 of the genomic DNA sequence shownin FIG. 3, with a substitution at nucleotide 252 (encoding a Gly atresidue 23). Plasmid pHAG1 has been deposited with American Type CultureCollection under accession number 40192.

A human genomic library (Maniatis et al., Cell 15: 687-702, 1978)consisting of about 3×10⁶ λ Charon 4A bacteriophage was screened withthe cDNA insert of clone pHAG1 which had been radiolabeled by nicktranslation (Rigby et al., J. Mol. Biol. 113: 237-251, 1977). Onestrongly hybridizing phage clone, designated λHAG1, identified by themethod of Benton & Davis (Science 196: 181-182, 1977) was plaquepurified, and the phage DNA was isolated by the plate lysis method(Maniatis et al., 1982, ibid). The genomic insert was analyzed bydigestion with various restriction enzymes. A DNA fragment generated bydigestion of the insert with PvuII was about 5 kilo bases in size andstrongly hybridized to the cDNA probe. This fragment was subcloned intoplasmid pBR322 and subjected to DNA sequencing by the dideoxy method(Messing et al., Nucleic Acids Res. 9: 309-321, 1981; Norrander et al.,Gene 26: 101-106, 1983) employing [³⁵ S]dATPαS as described in theAmersham cloning and sequencing manual. A ˜3 kb DNA fragment generatedby digestion of the phage genomic insert with KpnI which stronglyhybridized to the probe was subcloned into the M13mp18 phage vector andsubjected to DNA sequencing employing the synthetic oligonucleotideprobe as a primer. Systematic deletions of the genomic DNA withendonuclease Bal 31 were carried out essentially as described by Ponczet al. (Proc. Natl. Acad. Sci. USA 79: 4298-4302, 1982), Guo & Wu (Meth.in Enzymology 100: 60-96, 1983). About 95% of the genomic DNA sequencewas determined two or more times, and greater than 50% of the genomicsequence was determined on both strands. A portion of this sequencecorresponded to the coding sequence for angiogenin. The vector λHAG1 hasbeen deposited with American Type Culture Collection under accessionnumber 40193.

The gene for angiogenin was also found in a DNA fragment of about 5 kilobases that was generated by digestion of λHAG1 with PvuII. This DNAfragment was subcloned into pBR322 and subjected to DNA sequencing bythe dideoxy chain termination method employing the strategy shown in thebottom of FIG. 2. The complete sequence of the gene for human angiogenin(FIG. 3) indicated that the gene contains about 800 nucleotides and isfree of intervening sequences in the coding and 3' noncoding regions ofthe gene. The possibility of an intron(s) in the 5' flanking regioncannot be excluded, however, since the largest cDNA did not extend intothis region.

The gene for angiogenin contains three Alu repetitive sequences (Schmid& Jelinek, Science 216: 1065-1070, 1982) in its flanking regions (FIGS.2 and 3). The first Alu repeat was located in the immediate 5' flankingregion of the gene, while the second was present in the immediate 3'flanking region. These two Alu repeats were in the same invertedorientation. The third Alu repeat was located about 500 nucleotidesdownstream from the second Alu sequence in the 3' flanking region of thegene and was in the typical orientation with the poly(A) on the 3' endof the 300 nucleotide sequence. Furthermore, each Alu repeat was flankedby a pair of short direct repeat sequences. The nucleotide sequences forthe three Alu repeats were about 87% homologous to the consensus Alusequence of Schmid & Jelinek (ibid).

A tentative TATA box (Goldberg, M. L., Doctoral Dissertation, StanfordUniversity, 1979) and a transcription initiation site were identified atnucleotides -32 and +1 respectively, but no potential CAAT box was foundin the immediate vicinity. A sequence of TCAAT was identified, however,at nucleotide -255 which is about 190 bp upstream from the proposed TATAbox. Two sequences of AATAAA which are involved in the polyadenylationor processing of the messenger RNA at the 3' end (Proudfoot & Brownlee,Nature 263: 211-214, 1976), were identified at nucleotides 703 and 707.Polyadenylation of the mRNA occurs at nucleotide 731 which is 20nucleotides downstream from the end of the second AATAAA sequence. Theconsensus sequence of CACTG, which also may be involved inpolyadenylation or cleavage of the mRNA at the 3' end (Berget, Nature309: 179-182, 1984), was present starting at nucleotide 747. A stretchof 32 nucleotides with alternating purine and pyrimidine was foundstarting with nucleotide 1416. This sequence provides a potential regionfor a left-handed helix structure or Z-DNA in the gene Rich et al., Ann.Rev. Biochem. 53: 791-846, 1984). A computer search of the flankingregions of the gene for angiogenin as well as in the complementarystrand showed no open reading frames.

The amino acid sequence of human angiogenin is about 35% homologous withhuman ribonuclease. The location of the disulfide bonds, determined bydirect protein sequence analysis, further emphasizes the homology toribonuclease.

EXAMPLE 2 Production of Angiogenin in Transfected Mammalian Cells

For expressing angiogenin in transfected mammalian cells, expressionvector pHAGF-MT-DHFR, comprising the angiogenin genomic coding sequence(HAGF), the mouse metallothionein-1 (MT-1) promoter, and a DHFRselectable marker joined to the SV40 promoter, was constructed.

As shown in FIG. 4, the HAGF insert was isolated from λHAG1 as a PvuIIfragment and inserted into pBR322 which had been linearized with SmaI.The resultant plasmid was then digested with Bg1II, which cuts in the 5'untranslated region of the HAGF sequence. The DNA was then digested withBal 31 to produce a HAGF sequence having a 5' terminus at nucleotide +7from the site of transcription initiation. The DNA was then digestedwith BamHI. The resulting fragment ends were blunted using DNApolymerase I (Klenow fragment) and the fragment comprising the pBR322and HAGF coding sequences was purified by electrophoresis on a 0.7%agarose gel. The DNA was extracted from the gel and recircularized. Theresultant plasmid, designated pBR322-HAGF, was digested with BamHI andEcoRI and the ˜3 kb fragment comprising the angiogenin sequence waspurified by electrophoresis on a 0.7% agarose gel.

The final expression vector was then constructed in the followingmanner. Plasmid pMTFIX (Kurachi and Palmiter, Thrombosis and Hemostasis54: 282, 1985), comprising the mouse metallothionein (MT-1) promoter,human Factor IX coding sequence, SV40 promoter, and a modified DHFR gene(Levinson et al., EPO publication 117,060) was digested with BamHI andEcoRI (FIG. 4). The fragment comprising the pUC13 sequence and theSV40-DHFR expression unit was gel purified. This segment was then joinedto the BamHI-EcoRI HAGF fragment. The resultant vector was designatedpHAGF-MT-DHFR (FIG. 4).

Plasmid pHAGF-MT-DHFR was then transferred into baby hamster kidney(BHK) cells by standard calcium phosphate-mediated transfectionprocedures. Cells containing the vector were grown at 37° C. in 5% CO₂in Dulbecco's modified Eagle's medium containing glucose and glutamine(Gibco), supplemented with 3.7 g/l NaHCO₃, 10% heat inactivated fetalcalf serum and antibiotics. Cells containing the plasmid were thenselected for methotrexate (MTX) resistance by sequentially increasingthe concentration of MTX in the culture medium. MTX concentrations usedwere 1 μM, 10 μM, 100 μM, and 1 mM. Cells which survived in the presenceof 1 mM MTX were then induced by the addition of either 80 μM ZnSO₄, 2μM CdSO₄, or a mixture of the two salts to the culture medium.

Angiogenin mRNA was assayed essentially as described by Durnam andPalmiter (Analyt. Biochem. 131: 385-393, 1983). Sense strand DNA from anM13mp18 clone containing the entire angiogenin gene in a ˜2.9 kb insertwas used to make a standard curve. A dodecaoligonucleotide complementaryto the coding sequence for amino acids 35 to 41 of angiogenin waslabelled with ³² P at its 5' end and used as a probe in solutionhybridization.

Messenger RNA levels were elevated>20-fold using Cd⁺⁺ inductionand>15-fold for Zn⁺⁺ induction.

To assay for the presence of angiogenin, the induced, conditioned mediumwas acidified, frozen and thawed, centrifuged, and the supernatantdialyzed against water and lyophilized. The lyophilized material wasthen dissolved in and dialyzed against 0.1M sodium phosphate buffer pH6.6, supplemented with lysozyme as a carrier. The dialyzed sample wasapplied to a column of CM-52 cellulose and partially purified angiogeninwas eluted with the same buffer containing 1M NaCl. The eluate wasapplied to a C18 reversed phase HPLC column and fractionated asdescribed above. A protein having the chromatographic andelectrophoretic properties of tumor-derived angiogenin was obtained.

The resultant protein is assayed for angiogenic activity by the CAMmethod using published procedures.

EXAMPLE 3 Production of Angiogenin in Yeast

A vector for expressing angiogenin in transformed yeast is illustratedin FIG. 6. It contains an expression unit consisting of the yeast ADHIIpromoter (Young et al., in Genetic Engineering of Microorganisms forChemicals, Hollaender et al., eds., p. 335, Plenum, New York, 1982), aportion of the MF α1 pre-pro sequence (Kurjan and Herskowitz, Cell 30:933-943, 1982), and the HAGF sequence.

A portion of the ADHII gene is obtained from the plasmid pADR2 (Beierand Young, Nature 300: 724-728, 1982) as a SphI fragment ofapproximately 1530 bp. This fragment is subcloned into an M13 phagevector and mutagenized essentially as described by Zoller et al. (Manualfor Advanced Techniques in Molecular Cloning Course, Cold Spring HarborLaboratory, 1983) using a mutagenic primer having the sequence GTA ATACAC AGA ATT CAT TCC AGA AA. The replicative form of the mutagenizedphage is digested with SphI and Eco RI and a partial ADH II promoterfragment of approximately 176 bp is purified. The upstream portion ofthe promoter is then restored by joining the ˜176 bp fragment, the ˜1 kbBam HI-SphI fragment of ADH II (from pADR2), and Bam HI+Eco RI cutpUC13. The resultant plasmid is designated pUCADH2 (FIG. 6).

Referring to FIG. 5, the MFα1 gene is obtained from a yeast genomiclibrary of partial Sau 3A fragments cloned into the Bam HI site of YEp13(Nasmyth and Tatchell, Cell 19: 753-764, 1980) and identified bycomplementation of the matα2 mutation. One such clone is designatedpZA2. The MFα1 sequence is cut at position -71 with HinfI, the endsfilled using DNA polymerase I (Klenow fragment), and Eco RI linkers areadded. The signal sequence is then isolated as an Eco RI-Hind IIIfragment and subcloned in pUC12 to construct plasmid pUCPPαF.

The HAGF coding sequence is isolated from λHAG1 as a 1115 bp AccIfragment. The fragment ends are blunted using DNA polymerase I (Klenowfragment) and Hind III linkers are added to the ends. The resultantfragment is digested with Hind III and Eco RV and a ˜666 bp fragment isgel purified. Sal I linkers are then ligated to the Eco RV terminus, thefragment is cut with Sal I, and the ˜674 bp fragment is gel purified.

The HAGF sequence is then joined to a portion of the MFα1 signalsequence. Plasmid pUCPPαF is digested with PstI and Hind III and the 237bp fragment is isolated. The ˜674 bp HAGF fragment and the 237 bp MFα1fragment are ligated to Pst I+Sal I cut pUC13. The resultant recombinantplasmid is digested with Pst I and Sal I and the ˜911 bp MFα1-HAGFfragment is gel purified and inserted into Pst I+Sal I cut M13mp10(replicative form). A precise junction between the Lys-Arg processingsite of MFα1 and the first amino acid of angiogenin is achieved throughin vitro mutagenesis of the resultant recombinant phage using themutagenic primer TGG ATA AAA GAC AGG ATA ACTC. The replicative form ofthe mutagenized phage is cut with Pst I and Sal I and the ˜880 bpMFα1-HAGF fragment is gel purified.

Referring to FIG. 6, the ADH II-MFα1-HAGF expression unit is thenassembled. Plasmid pUCADH2 is cut with Bam HI and Eco RI an the ˜1200 bpADH II fragment is gel purified. Plasmid pUCPPαF is cut with Eco RI andHind III and the ˜340 bp MFα1 fragment is gel purified. The twofragments are ligated to Bam HI+Hind III cut pUC12 to constructpUCADHPP. This plasmid is digested with Bam HI and Pst I and the ˜1300bp ADHII-MFα1 fragment is purified. This fragment and the ˜880 bpMFα1-HAGF fragment are then joined, in a triple ligation, to Bam HI+SalI cut pUC12. The resultant plasmid is designated pUCAMA.

The yeast expression vector pYAGF is constructed by ligating the BamHI-Hind III expression unit fragment from pUCAMA to Bam HI+Hind IIIdigested YEp13.

Yeast cells are transformed with pYAGF and cultured by conventionalmethods. Angiogenin is purified from cell extracts or culture mediaessentially as described above.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

We claim:
 1. A plasmid identical to ATCC Deposit No.
 40192. 2. Apurified isolated DNA sequence consisting essentially of a DNA sequencecoding for the protein defined in FIG. 1 or for a protein havingsubstantially the same amino acid sequence and substantially the sameangiogenic activity as the protein defined in FIG.
 1. 3. A plasmididentical to ATCC Deposit No.
 40193. 4. An isolated vector forexpressing in transformed yeast the protein defined in FIG. 1 or aprotein having substantially the same amino acid sequence andsubstantially the same angiogenic activity as the protein of FIG. 1,which vector includes a DNA construct comprising the DNA sequenceclaimed in claim 2, a promoter upstream of and operably linked to saidsequence, and a polyadenylation signal downstream thereof.
 5. A vectoras claimed in claim 4 in which said promoter is the yeast ADHIIpromoter.
 6. An isolated vector for expressing in a transformedmammalian cell the protein defined in FIG. 1 or a protein havingsubstantially the same amino acid sequence and substantially the sameangiogenic activity as the protein of FIG. 1, which vector includes aDNA construct comprising the DNA sequence claimed in claim 2, a promoterupstream of and operably linked to said sequence, and a polyadenylationsignal downstream thereof.
 7. A vector as claimed in claim 6 in whichsaid promoter is the mouse metallothionein-1 promoter.
 8. A yeast cellcontaining the DNA sequence coding for the protein defined in FIG. 1 ora protein having substantially the same amino acid sequence andsubstantially the same angiogenic activity as the protein of FIG. 1, apromoter upstream of and operably linked to said sequence, and apolyadenylation signal downstream thereof.
 9. A method for producing theprotein defined in FIG. 1 or a protein having substantially the sameamino acid sequence and substantially the same angiogenic activity asthe protein of FIG. 1, comprising the steps of inserting into cells aDNA vector having a construct comprising the DNA sequence claimed inclaim 2, a promoter upstream of and operably linked to said sequence,and a polyadenylation signal downstream thereof, growing said cells in asuitable medium, and separating said protein from said cells.
 10. Amethod as claimed in claim 9 wherein said cell is a yeast cell.
 11. Amethod as claimed in claim 9 wherein said cell is a mammalian cell. 12.A method as claimed in claim 9 wherein said cell is a bacterial cell.